Fabrication of living blood vessels and glandular tissues

ABSTRACT

A method and apparatus for producing a vessel-equivalent prosthesis is described. A contractile agent such as fibroblast cells, smooth muscle cells or platelets is incorporated into a collagen lattice and contracts the lattice axially around an inner core. After the structure has set, additional layers may be formed in an ordered manner depending on the intended function of the prosthesis. Alternatively, all the layers may be formed concurrently. A plastic mesh sleeve is sandwiched between layers or embedded within the smooth muscle cell layer to reinforce the structure and provide sufficient elasticity to withstand intravascular pressure.

RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 261,928 filed May8, 1981, now U.S. Pat. No. 4,539,716 and is related to Ser. No. 972,832filed Dec. 26, 1978 and Ser. No. 245,536 filed Mar. 19, 1981, both ofwhich are now abandoned in favor of continuation-in-part applications.

TECHNICAL FIELD

This invention is in the field of biology and particularly relates tothe fabrication of living tissue in tubular form for variousapplications such as capillaries, larger blood vessels and glandularprosthesis.

BACKGROUND ART

Some of the material in the first of the referenced related applicationsabove has been published in the Proc. Natl. Acad. Sci. USA Vol. 76 No. 3pp 1274-1278 March 79 in an article entitled "Production of aTissue-Like Structure by Contraction of Collagen Lattices by HumanFibroblasts of Different Proliferative Potential In Vitro" by Bell etal. This article and the related applications are mainly concerned bythe fabrication of planar surfaces of skin-like living tissue. Thisliving tissue is produced in vitro by forming a hydrated collagenlattice, containing a contractile agent, such as fibroblast cells orblood platelets which contract the lattice. This skin-like tissue isformed in a round or rectangular vessel with, or without, a frame ofstainless steel mesh lying on the floor of the vessel. In its absence,the lattice contracts in all dimensions; in its presence as the latticesets it becomes anchored to the mesh and contracts in the thicknessdimension only. The mesh, resembling a picture frame, holds the latticeof living tissue within it. The contracted lattice, with or without thestainless steel mesh frame, can be seeded with epidermal cells from thepotential graft recipient. When a sheet of epidermal cells forms, thetwo layered skin equivalent is grafted.

The resultant graft is unique as compared to any other graft obtainedfrom artificial skin since its basic organization is like that of skinand its living constituent cells are donated by potential graftrecipients.

DISCLOSURE OF THE INVENTION

This invention relates to the casting of living collagen latticescontracted by living cells, such as fibroblasts, smooth muscle cells, orelements of cells such as blood platelets. In particular, the latticesare cast into shapes which provide internal surface areas and tubularshaped terminals, or end structures, particularly effective for makingconnections, in vivo, with existing tubular structures, such ascapillaries, blood vessels and glandular tissues.

The internal surface of the cast structure is lined with specializedcells, depending on the function of the structure. For example,endothelial cells are used for the internal surface of an artery, vein,or other structures with internal surfaces.

Alternatively, in some applications it may be desirable to line theinternal surface with specialized cells having a predeterminedtherapeutic value. For example, the inner surfaces of a capillary bedmay be lined with pancreatic β cells to boost the insulin supply in theblood. Pancreatic islets (islets of Langerhans), hepatocytes or othertypes of glandular cells may also be used for lining the inner surfaceof the vessel-equivalent structures.

In one embodiment, the structure is in the form of a tube, or cylinder.The central core for forming the tube consists of polyethelene or glasstubing. This core is axially centered within a cylindrical mold.Suitable tissue forming constituents are poured into the cylindricalmold. After a suitable period of time, the tissue forming constituentscontract the lattice and close in around the central core. Thisprocedure can be repeated as many times as desired with the same ordifferent cell types in the same or different proportions to yield amultilayer tube. After each layer contracts the fluid expressed from thecontracting lattice is poured off to accomodate the tissue formingconstituents of the next layer. The central core may then be removed andsuitable cells, predicated on the function of the cast structure, maythen be cultured on the inner surface of the hollow tissue cylinders, toform, for example, a vessel-equivalent structure.

The fortuitous fact that the lattice contracts radially about thecentral core structure to form tubes enables one to form various shapedstructures defined by the inner core surface. If, instead, the latticecontracted in all directions, the resultant structure would end up as ashapeless mass at the bottom of the mold. It is also important to notethat in the formation of vessel-equivalent structure, in accordance withthe invention, the sequential addition of cells in an ordered pattern oflayers is essential.

The vessel-equivalent structure thus far described is devoid of elastin,the fibrous mucoprotein which is the major connective tissue protein ofelastic structures (e.g. large blood vessels). Without this elasticproperty it is possible that the vessel could burst under pressure.Since elastin is an extremely insoluble substance it is difficult todirectly incorporate elastin into the molded tissue forming constituentspreviously described. Accordingly, a plastic mesh may be optionallyprovided between two layers or within a layer of the tissue formingconstituents during the molding process, as will be described in detail.

This mesh serves to reinforce the resultant vessel and at the same timeprovide a degree of elasticity to the structure so that it may expandand contract in the manner of a natural blood vessel having elastin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a first embodiment of the inventionshowing the structure of the casting chamber.

FIG. 1A is a cross-sectional view of FIG. 1 showing a vessel as cast.

FIG. 1B is a perspective view showing a plastic mesh on a support tubewhich is used to position the mesh during casting.

FIG. 2 is a schematicized view showing the culturing apparatus of theinvention.

BEST MODE OF CARRYING OUT THE INVENTION

The following description generally relates to the casting ofcylindrical structures intended as prosthesis for vessels or capillariessince such structures are commonly found in the human body. However,other shapes may be conviently cast in accordance with the teachingsherein and the invention is not intended to be limited to any particularshape or body structure.

FIG. 1 shows a preferred form of casting chamber for fabricating a bloodvessel-equivalent of living matter. The casting chamber 10 comprises acentral rod or mandrel 12 disposed in a cylinder 16. The central rod andcylinder are mounted on a base or stand 14. The rod 12 is provided withthree arms or spokes 18 at the top of the rod for centering the rodwithin the cylinder 16.

The base is provided with an appropriate collar 20 to accept the centralrod 12. The outer cylinder has an internal diameter such that when thearms 18 are disposed as shown and the central rod is located in thecollar 20, the rod 12 will be centered within cylinder 16. The outerdiameter of the rod 12 determines the inner diameter of the cast vesseland for many applications would be in the range of from 2-10 mm.

With the diameter of the central rod kept constant, the inner diameterof cylinder 16 will determine the final thickness of the cast layer, andtypically may range from 1-4 cm to produce a final thickness of about0.5-2 mm, the final thickness being proportional to the diameter. Theheight of the chamber determines the length of the vessel and wouldtypically be between 10-30 cm in height.

The casting chamber parts should be made from material which may bereadily cleaned and is autoclavable. Preferably, the cylinder 16 shouldbe made from material which is clear and which will permit diffusion ofcarbon dioxide and other gases. Thus, the rod 12 may be made of glass ormetal and the cylinder 16 should preferably be made of autoclavableplastic, such as polycarbonate. The stand 14 may be made of glass,plastic or metal, such as stainless steel.

The size and structure of blood vessels varies in accordance with thefunction of the particular blood vessel. Blood vessels may be generallycharacterized by their cellular composition and the composition of thematrix or collagen lattice with which other extracellular elements, suchas elastin fibers and proteoglycans are associated. The collagen,elastin, and proteoglycans are the biosynthetic products of the cells ineach of the layers.

The cell types are endothelial, smooth muscle, and fibroblasts (calledpericytes) and are found respectively in successive layers from thelumen outward. In order to construct a particular type of blood vessel,the respective layers may be laid down in order. Alternatively, severalcan be laid down concurrently. All vessels contain an inner endotheliallining. In an artery, for example, smooth muscle surrounds theendothelium and the final outside layer is made up of fibroblasts.

The process for fabricating the above described multilayered bloodvessel-equivalent will now be described in detail in connection withFIGS. 1 and 2.

First, the smooth muscle layer is fabricated. A mixture of nutrientmedium (e.g. McCoy's medium containing fetal bovine serum) is preparedin a flask. The ingredients are mixed in the following ratio: 9.2 ml of1.76×concentrate of McCoy's medium and 1.8 ml of fetal bovine serum. ThepH is raised by addition of 1.0 ml of 0.1N NaOH. The foregoing mixtureof medium and serum is poured onto a dish in which 1.5 ml of nativecollagen in a 1-1000 acidic acid solution has been prepared. About250,000 cultured aorta smooth muscle cells suspended in a 0.5 ml ofMcCoy's medium supplemented with a 10% fetal bovine serum is quicklyadded. The above constituents are mixed by swirling the dish and quicklypouring the mixture into the casting chamber. The chamber is then placedin a humidified 5% CO₂, 95% air incubator at 37° C. for 3 days.

A collagen lattice or gel forms immediately on casting the mixture. Thecollagen fibrils are gradually compacted by the cells so that fluid issqueezed out of the lattice. The result is contraction of the collagenlattice around the central core or rod 12. After 3 days in theincubator, the smooth muscle layer will have set in a cylindricalstructure having sufficient structural integrity to simulate, orreplicate, the smooth muscle layer of a typical blood vessel. If asecond layer is to be applied, the fluid expressed during contraction ofthe first lattice is poured off and a second complete mixture of allingredients is added to replace the fluid. The process may be repeatedas many times as desired to give a multilayered structure. The layersmay be poured simultaneously with a removable separation or sleeve (notshown) between them. As soon as gelation begins the sleeve is removed.

Optionally, after the smooth muscle layer cylinder has been cast, it maybe desirable to provide a plastic mesh sleeve 11 about the outer surfaceof the smooth muscle layer cylinder or the mesh may be embedded in thesmooth muscle layer. This mesh will serve to reinforce the resultingstructure and provide some degree of elasticity so that the resultingstructure will be better able to withstand the pressures it will besubjected to in use. Meadox Medicals, Inc., 103 Bauer Drive, Oaklane,N.J. 07436, supplies a Dacron® mesh sleeve, Part No. 01H183, which hasproved particularly suitable for this purpose. Other suitable meshes arereadily available in various inert plastics, such as Teflon®, nylon,etc. and the invention is not to be limited to a particular plasticmaterial. Preferably, the mesh should be treated to render it moreelectronegative by, for example, subjecting it to plasma. This resultsin better cell attachment to the plastic sleeve and hence an increase inthe strength of the resultant structure.

The sleeve 11 should be placed on the smooth muscle cell cylinder byfirst disposing the sleeve 11 on metal tube 15 (as shown in FIG. 1B)which has an inner diameter larger than the outer diameter of the smoothmuscle cell cylinder. The tube 15, with the sleeve on the exterior, isthen slipped over the smooth muscle cell cylinder, a portion of thesleeve is then pulled off the tube 15 and onto the smooth muscle cellcylinder and held there while the tube 15 is slipped off the smoothmuscle cell cylinder. This procedure minimizes damage to the exteriorsurfaces of the smooth muscle cell cylinder while attaching the sleeve.

Next, a fibroblast layer may be cast around the inner smooth musclelayer(s) and sleeve 11 so as to completely enclose the sleeve 11, asshown in FIG. 1A. In this process, the ingredients described above inconnection with the fabrication of a smooth muscle layer are used toconstitute a fibroblast layer, except that cultured aorta fibroblastsare substituted for the smooth muscle cells. The incubation period forthe fibroblast layer may be 2 days to a week.

The resultant multi-layered structure consisting of inner smooth musclelayer(s) and an outer fibroblast layer with a mesh sleeve sandwichedbetween the two layers is now ready to be cultured with an innerendothelial lining of living endothelial cells. To perform this step thecylindrical tissue tube of several layers is slipped off the casting rod12 to receive the endothelial cells as a suspension. It is supported inthe culturing apparatus shown in FIG. 2.

The apparatus of FIG. 2 comprises a transparent chamber 24, within whicha rotatable rod 26 is inserted at one end and a rotatable tube 36 isinserted at the opposite end. The tube 36 and rod 26 are tied togetherby wire frame member 30 such that when the rod 26 is rotated, the tube36 will rotate in unison in the same direction. Rod 26 is coupled tomotor 28 such that when motor 28 is energized the rod 26 will rotate inthe direction shown by the arrow. Preferably, the rod is attached to themotor in such a way that the length of the rod inserted into the chamber24 may be adjusted in accordance with the length of thevessel-equivalent 44 being supported within the culture chamber 24. Thismay be accomplished by a rack and pinion device or other such variablelength means (not shown).

Rod 26 is provided at one end with a nipple 32 to which a vessel 44(such as the structure previously described in connection with FIGS. 1,1A and 1B comprising an inner cylinder smooth muscle cell layer, and anouter cylinder of fibroblast cells with a mesh sleeve sandwichedbetween) may be attached. Similarly, tube 36 is provided with acomplementary nipple 34 to which the opposite end of the vessel 44 maybe attached. In this manner, the vessel 44 is suspended between the rod26 and tube 36 and a culture medium may be introduced from reservoir 42through tubing 40 and fixed connecting tube 38, through tube 36 and intothe interior lining of blood vessel-equivalent 44. It should beunderstood that water-tight seal bearings (not shown) are provided atboth ends of chamber 24 to permit the rod and tube to be inserted intothe chamber.

Reservoir 42 is supplied with a suspension of about 200,000 culturedaorta or other endothelial cells in McCoy's medium supplemented with a20% fetal bovine serum. This mixture is fed by hydrostatic pressure fromthe reservoir into the vessel 44 as previously mentioned. Next, thevessel 44 is slowly rotated by means of motor 28 which preferably runsat a speed of between 0.1 and 1 r.p.m. Rotation of the vessel 44 enablesdistribution of the endothelial cells evenly on the inner lining of thevessel and the hydrostatic pressure head from the reservoir enables thelumen, or inner opening, of the vessel-equivalent to remain open. Itshould be emphasized that the above procedures are intended to becarried out asceptically.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, other equivalents for the specificreactants, steps and techniques, etc. described herein. Such equivalentsare intended to be included within the scope of the following claims.

I claim:
 1. In a method of producing, in vitro, a living multi-layeredtubular structure, comprising:a. producing a first tubular layer by:(1)forming a first aqueous mixture of collagen fibrils, nutrient medium anda first cellular contractile agent capable of interacting with collagenfibrils; (2) introducing said first mixture into an annular castingchamber; (3) maintaining the annular casting chamber containing saidfirst mixture under conditions sufficient to allow a gel to form thereinand to allow radial contraction of the gel with expression of aqueousliquid therefrom resulting from interaction of the cellular contractileagent with collagen fibrils to thereby form a contracted hydratedcollagen lattice suitable as one layer of a multi-layered tubularstructure; and (4) removing aqueous liquid expressed in the formation ofsaid first tubular layer from the annular casting chamber; b. producinga second tubular layer outwardly of said first tubular layer by:(1)forming a second aqueous mixture of collagen fibrils, nutrient mediumand a second cellular contractile agent capable of interacting withcollagen fibrils; (2) introducing said second mixture into the annularcasting chamber; (3) maintaining the annular casting chamber containingsaid second mixture under conditions sufficient to allow a gel to formtherein and to allow radial contraction of the gel with expression ofaqueous liquid therefrom resulting from interaction of the cellularcontractile agent with collagen fibrils to thereby form a contractedhydrated collagen lattice suitable as another layer of a multi-layeredtubular structure; and c. removing said multi-layered tubular structurefrom the annular casting chamber;The improvement comprising adding areinforcing sleeve of inert material to said living multi-layeredtubular structure.
 2. The improvement of claim 1 wherein saidreinforcing sleeve of inert material comprises a plastic mesh sleeve. 3.The improvement of claim 2 wherein said plastic mesh sleeve is locatedbetween the first and second tubular layers of the living multi-layeredtubular structure.
 4. The improvement of claim 3 wherein the plasticmesh sleeve is attached by first slipping it on a tube and subsequentlysliding the tube over the first tubular structure and removing the tubeleaving the sleeve positioned over the first tubular structure.
 5. Theimprovement of claim 3 wherein said plastic mesh sleeve is pretreated torender it more electronegative.
 6. The improvement of claim 5 whereinsaid plastic mesh screen is pretreated by subjecting it to plasma.
 7. Aliving multi-layered tubular structure produced by the improvement ofclaim
 1. 8. A living multi-layered tubular structure produced by theimprovement of claim
 3. 9. In a method of producing a living prosthesis,in vitro, comprising the steps of:a. fabricating a cylindrical smoothmuscle cell layer as follows:(aa) separately preparing (i) an aqueousacidic mixture comprising nutrient medium and collagen fibrils and (ii)a mixture of smooth muscle cells suspended in nutrient medium; (ab)raising the pH of mixture (i) and quickly combining mixture (i) andmixture (ii) and pouring the combined mixture into a casting chamberhaving an inner core member and an outer cylindrical wall structure toform a lattice; (ac) incubating the lattice for a period sufficient toenable collagen fibrils to be compacted by the cells so that aqueousliquid is expressed out of the lattice as the lattice contracts radiallyabout the core; (ad) removing aqueous liquid expressed in step (ac);(ae) repeating steps (aa)-(ad) if additional layers of smooth musclecells are desired; b. fabricating a layer containing fibroblast cells onsaid cylindrical smooth muscle layer as follows:(ba) separatelypreparing (i) an aqueous acidic mixture comprising nutrient medium andcollagen fibrils and (ii) a mixture of fibroblast cells suspended innutrient medium; (bb) raising the pH of mixture (i) and quicklycombining mixture (i) and mixture (ii) and pouring the combined mixtureinto a casting chamber having as an inner core member of cylindricalsmooth muscle cell layer and an outer cylindrical wall structure to forma lattice; (bc) incubating the lattice formed in step (bb) in accordancewith step (ac); (bd) removing aqueous liquid expressed in step (bc); c.lining the inner wall of the cylindrical smooth muscle cell layer withliving cells;The improvement of including a plastic mesh within saidliving prosthesis to thereby reinforce said living prosthesis and toprovide it with an increased degree of elasticity.
 10. The improvementof claim 8 wherein said plastic mesh comprises a sleeve positionedbetween the layers of smooth muscle cells and fibroblast cells.
 11. Theimprovement of claim 10 wherein said plastic mesh screen comprisespolyethylene terephthalate.
 12. The improvement of claim 11 wherein saidplastic mesh screen is pretreated to make it more electronegative. 13.The improvement of claim 12 wherein said pretreatment is done byexposing the plastic mesh screen to plasma.
 14. A living multi-layeredtubular structure produced by the improvment of claim
 9. 15. In atubular prosthesis formed of multiple layers of hydrated collagenlattices contracted with living cells:The improvement comprisingincluding a plastic mesh sleeve embedded in the layers of saidprosthesis to provide reinforcement and elasticity thereto.
 16. Theimprovement of claim 15 wherein said plastic mesh sleeve is formed frompolyethylene teraphthalate.